The T-body approach: redirecting T cells with antibody specificity.

"T-bodies" are genetically engineered T cells armed with chimeric receptors whose extracellular recognition unit is comprised of an antibody-derived recognition domain and whose intracellular region is derived from lymphocyte stimulating moiety(ies). The structure of the prototypic chimeric receptor, also known as a chimeric immune receptor, is modular, designed to accomodate various functional domains and thereby to enable choice of specificity and controlled activation of T cells. The preferred antibody-derived recognition unit is a single chain variable fragment (scFv) that combines the specificity and binding residues of both the heavy and light chain variable regions of a monoclonal antibody. The most common lymphocyte activation moieties include a T-cell costimulatory (e.g. CD28) domain in tandem with a T-cell triggering (e.g. CD3zeta) moiety. By arming effector lymphocytes (such as T cells and natural killer cells) with such chimeric receptors, the engineered cell is redirected with a predefined specificity to any desired target antigen, in a non-HLA restricted manner. Chimeric receptor (CR) constructs are introduced ex vivo into T cells from peripheral lymphocytes of a given patient using retroviral vectors. Following infusion of the resulting T-bodies back into the patient, they traffic, reach their target site, and upon interaction with their target cell or tissue, they undergo activation and perform their predefined effector function. Therapeutic targets for the T-body approach include cancer and HIV-infected cells, or autoimmune effector cells. To date, the most investigated area is cancer therapy. Here, the T-bodies are advantageous because their tumor recognition is not HLA-specific and, therefore, the same constructs can be used for a wide spectrum of patients and cancers.

ErbB2 is a tumor associated antigen and a suitable therapeutic target in Wilms tumor.

PURPOSE: The modern multimodality therapeutic approach to Wilms tumor (WT), combining surgery with radiotherapy and chemotherapy results in high cure rates even for high stage disease. However, the combination of radiotherapy and chemotherapy is associated with severe early and late complications such as neutropenic sepsis, growth retardation and secondary malignancies. Therefore, novel therapeutic strategies, which would decrease the treatment burden, are required. We studied the expression of erbB2 growth factor receptor in WT cells as well as its role as a tumor therapeutic target in an in vivo xenograft model of Wilms tumor. MATERIALS AND METHODS: Paraffin embedded pathological samples from 14 different WT cases as well as xenografts derived thereof were immunostained with anti-erbB2 monoclonal antibody. The immunostaining was graded in comparison to a known positive control (breast cancer) and was scored by the intensity of staining (0 to +3) multiplied by the percentage of cells expressing the antigen. The expression of erbB2 in the human WT xenograft was verified also by fluorescence activated cell sorter analysis. In addition, nude mice bearing established subcutaneous human WT xenografts were treated with either 3 intraperitoneal injections of N29 anti-erbB2 monoclonal antibody or irrelevant antibody. RESULTS: All of the authentic human pathological samples, except 1 anaplastic WT as well the WT xenografts (at different stages), expressed erbB2. Expression was also observed in WT metastasis and in tumors which out grew chemotherapy. Systemic administration of anti-erbB2 monoclonal antibody inhibited and even prevented the growth of WT xenograft in vivo. CONCLUSIONS: ErbB2 is a tumor associated antigen in WT. Being expressed in almost all tumor stages, our in vivo model suggests that erbB2 may serve as a WT therapeutic target. Further work is needed to establish the role of erbB2 in the disease and its potential use in decreasing current treatment burden.

Functional expression of chimeric receptor genes in human T cells.

Tumor immunotherapy has been limited to date by the poor antigenicity of most tumors, the immunocompromised state of many cancer patients, and the slow tumor penetration and short half-life of exogenously-introduced anti-tumor antibodies. Our group has developed a model immunotherapy system using a chimeric construct containing an antibody V region fused to a T cell activation molecule (T body) introduced by transfection into cytotoxic T cell lines, or populations of activated primary T or natural killer (NK) cells. In this study we have optimized the conditions needed for efficient transduction of human peripheral lymphocytes (PBL) using retroviral vectors pseudotyped with the gibbon ape leukemia virus (GaLV) envelope. Selection of packaging cells producing high virus titers was performed following transfection with constructs containing the green fluorescent protein (GFP), and FACS sorting. As a model chimeric receptor gene we used a tripartite construct consisting of a single-chain anti-TNP antibody variable region linked to part of the extracellular domain and the membrane spanning regions of the CD28 coreceptor molecule and joined at its 5' end to a gene fragment encoding the intracellular moiety of the gamma activation molecule common to the Fcepsilon and Fcgamma receptors. Enriched preparations of retrovectors containing this chimeric receptor and the GFP gene could stably and efficiently transduce human PBL co-activated by anti-CD3 and anti-CD28 antibodies. In routine experiments, the transgene was expressed in 35-70% of the human T cells. Such lymphocytes express the chimeric receptors on their surface and upon stimulation with hapten immobilized on plastic they can produce IL-2. Transfectomas activated in this manner also undergo specific proliferation in the absence of exogenous IL-2. Moreover, the transduced lymphocytes could effectively lyse target cells expressing the TNP hapten on their surface. These studies establish the conditions for the optimal transfection of effector lymphocytes to redirect them against a variety of tumor targets.

WISH-PC2: a unique xenograft model of human prostatic small cell carcinoma.

Prostatic small cell carcinoma is an aggressive subtype of prostate cancer that usually appears as a progression of the original adenocarcinoma. We describe here the WISH-PC2, a novel neuroendocrine xenograft of small cell carcinoma of the prostate. This xenograft was established from a poorly differentiated prostate adenocarcinoma and is serially transplanted in immune-compromised mice where it grows within the prostate, liver, and bone, inducing osteolytic lesions with foci of osteoblastic activity. It secretes to the mouse Chromogranin A and expresses prostate plasma carcinoma tumor antigen-1, six-transmembrane epithelial antigen of the prostate, and members of the Erb-B receptor family. It does not express prostate-specific antigen, prostate stem cell antigen, prostate-specific membrane antigen, and androgen receptor, and it grows independently of androgen. Altogether, WISH-PC2 provides an unlimited source in which to study the involvement of neuroendocrine cells in the progression of prostatic adenocarcinoma and can serve as a novel model for the testing of new therapeutic strategies for prostatic small cell carcinoma.

Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR.

Primary human activated T lymphocytes were genetically grafted with chimeric T cell receptors (TCR). Three domain single chain (sc-) TCR as well as two chain (tc-) TCR gene constructs were derived from the melanoma-specific cytotoxic human T cell (CTL) clone 82/30, and linked to the CD3-zeta signaling element. Chimeric TCR alpha and beta receptor genes were structurally designed to prevent pairing with endogenous TCR alpha and beta chains in order to prevent the generation of unpredictable immune specificities. After transduction of polyclonally activated human peripheral blood lymphocytes with retroviral vectors harboring the chimeric receptor genes, genetically engineered cells specifically recognized and responded to MAGE-A1POS/HLA-A1POS cells. Importantly, each type of transduced T lymphocytes that bound specifically to peptide/MHC complexes also showed specific antitumor reactivity as well as lymphokine production. Genetically engineered primary human T lymphocytes expressing chimeric sc- or tc-TCR therefore hold promise for disease-specific therapies.

Recognition of human colon cancer by T cells transduced with a chimeric receptor gene.

Transduction with chimeric T-cell receptor genes can be used to redirect primary T lymphocytes to recognize specific antigens (Ags), including ovarian and breast cancer Ags. To extend this approach to colon cancer we report here redirection of T cells using a chimeric receptor recognizing the colon cancer-associated Ag EGP40. Chimeric T cell receptors were constructed by ligating single-chain genes of either of two EGP40-specific monoclonal antibodies (CO17.1 A, GA733) to the Fc receptor gamma-signaling chain. Retroviral vectors incorporating these constructs were used to transduce a murine T-cell line and human peripheral blood lymphocytes. These modified T cells were analyzed for specific recognition of colon cancer lines by measuring cytokine release and lytic activity against tumor targets. Murine lymphocytes transduced with the chimeric receptor based on GA733, but not CO17.1A, released cytokine specifically in response to EGP40-expressing colon cancer cell lines. Recognition of colon cancer targets by murine lymphocytes was blocked by the addition of GA733 antibody or soluble EGP40 Ag, confirming that colon cancer recognition is based on specific chimeric receptor-Ag interaction. Human lymphocytes transduced with chimeric GA733 specifically recognized colon carcinoma cells in cytokine release assays and lysed EGP40-expressing tumor cells. Genetic modification of T cells can be used to redirect T cells against EGP40-expressing tumor cells. The expression of chimeric GA733 in the autologous lymphocytes of patients may provide a source of tumor-reactive cells with therapeutic application against colon cancer.

Gene therapy approaches to HIV-infection: immunological strategies: use of T bodies and universal receptors to redirect cytolytic T-cells.

Combined regimens of classical antiviral treatments have not, until now, lead to the eradication of HIV-1. A specific anti-HIV immune response may have to be boosted or transferred to patients after suppression of viral replication, in order to eradicate residual infected cells from their sanctuaries. Cytotoxic T cells engineered to express recombinant chimeric receptors can be redirected against HIV-infected cells and could represent the basis of a new type of immunotherapy. Several HIV epitopes have been targeted successfully in vitro. Two types of binding domains (antibody fragments, CD4) fused with various signal transducing units (zeta chain of the CD3 complex, Fc epsilon RI gamma chain) have been tested for their ability to redirect effector cells to HIV infected lymphocytes. CD4-zeta-expressing myeloid and natural killer cells conferred SCID mice protection against challenge with tumor cells expressing HIV-env. Finally, the safety of the adoptive transfer of syngeneic CD4-zeta -modified T cells in HIV-infected individuals is currently under evaluation.

Conformational changes affect binding and catalysis by ester-hydrolysing antibodies.

D2.3, D2.4 and D2.5 are ester-hydrolysing antibodies raised against a phosphonate transition state analogue (TSA). All three antibody-TSA binding kinetics, as monitored by fluorescence quenching, indicate an "induced-fit" mechanism: fast bimolecular association followed by a unimolecular isomerisation (k=1-7 s-1). Isomerisation leads to a 30-170-fold increase in affinity towards the TSA and, consequently, to higher catalytic rates. Antibody D2.3 exhibits a complex three-step binding mechanism, in which the last step is a "very slow" isomerisation (k<0.02 s-1). This very slow isomerisation is limiting the rate of catalysis by D2.3, as indicated by the kinetics of product release which show characteristics of enzyme "conformational memory" or "hysteresis". The results support a mechanism consisting of pre-equilibrium between "nether-active" (low affinity) and "active" (high affinity) antibody conformers (prior to ligand addition) as well as induced-fit, i.e. isomerisation of the nether-active ligand-antibody complex to give the active complex. Crystal structures of these antibodies, free and complexed, have previously indicated that their conformation does not change upon binding. Here, we show that the buffer used to crystallise the antibodies, and in particular its polyethylene glycol component, alters the pre-equilibrium in favour of the active conformer, leading to its crystallisation both in the presence and in the absence of the TSA.

Loss of original antigenic specificity in T cell hybridomas transduced with a chimeric receptor containing single-chain Fv of an anti-collagen antibody and Fc epsilonRI-signaling gamma subunit.

T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted with a chimeric cell surface receptor, scC2Fv/gamma, which includes the single-chain Fv domain (scFv) of the anti-collagen type II mAb C2 and the signaling gamma subunit of the Fc epsilonRI. When transduced into MD.45 cells, scC2Fv/gamma or its mutated form lacking immunoreceptor tyrosine-based activation motif (ITAM), scC2Fv/gammaIC-, formed mainly homodimers. A small proportion of these molecules formed heterodimers with endogenous CD3zeta in these hybridoma cells. By contrast, in HCQ6 cells, the majority of scC2Fv/gamma and scC2Fv/gammaIC- molecules formed heterodimers with CD3zeta, and only a small proportion of them was expressed as homodimers. Stimulation with plastic-immobilized collagen induced IL-2 production in scC2Fv/gamma-transduced MD.45 cells, but not in MD.45 cells transduced with the ITAM-less chimera scC2Fv/gammaIC-. HCQ6 cells transduced with scC2Fv/gamma responded to plastic-bound collagen. Due to the high content of CD3zeta-associated chimeras, HCQ6 cells transduced with the ITAM-less scC2Fv/gammaIC- chimera were also responsive to plastic-bound collagen. When cells were stimulated with collagen in solution, MD.45 cells transduced with scC2Fv/gamma produced IL-2, whereas transduced HCQ6 cells were unresponsive, hence suggesting that the ability of cells transduced with scC2Fv chimeras to respond to soluble collagen correlated with predominant expression of divalent scC2Fv/gamma homodimers, but not monovalent scC2Fv/gamma-CD3zeta or scC2Fv/gammaIC(-)-CD3zeta heterodimers. Of interest, expression of CD3 subunits in hybridomas transduced with scC2Fv chimeras was reduced, resulting in decreased response to cognate Ags.

Crossreactivity, efficiency and catalytic specificity of an esterase-like antibody.

The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-nitrophenyl esters with significant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme specificity has been extensively studied by X-ray crystallography, structural data on catalytic antibodies have thus far related only to one of the reactions they catalyze. To investigate the substrate specificity and the substrate relative to product selectivity of D2.3, we have determined the structures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 A resolution, and that with p-nitrobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the structures suggest that a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic efficiency and substrate specificity. The structure of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 A resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located, and to the absence of a direct hydrogen bond between the product and the Fab. Catalytic specificity and the manner of product release are both affected by interactions with substrate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic antibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic efficiency incorporated in the procedure used to obtain it.

Targeting of interleukin 2 to human ovarian carcinoma by fusion with a single-chain Fv of antifolate receptor antibody.

To provide a new tool for the immunotherapy of human ovarian carcinoma, we constructed a fusion protein between interleukin-2 (IL-2) and the single-chain Fv (scFv) of MOV19, a monoclonal antibody directed against alpha-folate receptor (alpha-FR), known to be overexpressed on human nonmucinous ovarian carcinoma. This was accomplished by fusing the coding sequences in a single open reading frame and expressing the IL-2/MOV19 scFv chimera under the control of the murine immunoglobulin K promoter in J558L plasmacytoma cells. The design allowed the construction of a small molecule combining the specificity of MOV19 with the immunostimulatory activity of IL-2. This might improve the tissue penetration and distribution of the fusion protein within the tumor, reduce its immunogenicity, and avoid the toxicity related to the systemic administration of IL-2. The IL-2/MOV19 fusion protein was stable on purification from the cell supernatant and was biologically active. Importantly, this construct was able to target IL-2 onto the surface of alpha-FR-overexpressing tumor cells and stimulated the proliferation of the IL-2-dependent CTLL-2 cell line as well as that of human resting peripheral blood lymphocytes. In a syngeneic mouse model, IL-2/MOV19 scFv specifically targeted a-FR gene-transduced metastatic tumor cells without accumulating in normal tissues, due to its fast clearance from the body. Prolonged release of IL-2/MOV19 scFv by in vivo transplanted J558-EF6.1 producer cells protected 60% of mice from the development of lung metastases caused by an i.v. injection of a-FR gene-transduced tumor cells. Moreover, treatment with IL-2/MOV19 scFv, but not with recombinant IL-2, significantly reduced the volume of s.c. tumors. The pharmacokinetics and biological characteristics of IL-2/NMOV19 scFv might allow us to combine the systemic administration of this molecule with the adoptive transfer of in vitro retargeted T lymphocytes for the treatment of ovarian cancer, thereby providing local delivery of IL-2 without toxicity.

Harnessing Syk family tyrosine kinases as signaling domains for chimeric single chain of the variable domain receptors: optimal design for T cell activation.

T cells of tumor bearers often show defective TCR-mediated signaling events and, therefore, exhibit impaired immune responses. As such, patients with heavy tumor burden are often not amenable to adoptive T cell therapy. To overcome this limitation, we have developed a chimeric receptor that joins an extracellular single chain Fv (scFv) of a specific Ab for Ag recognition to an intracellular protein tyrosine kinase (PTK) for signal propagation. Stimulation through the scFv-PTK receptor should bypass defective TCR-proximal events and directly access the T cell's effector mechanisms. In this study we describe the optimization of a scFv-PTK configuration, leading to complete T cell activation. The cytosolic PTK Syk is superior to its family member, Zap-70, for intracellular signaling. As a transmembrane (TM) domain, CD4 performs better than CD8 when plastic-immobilized Ag serves as a stimulator. However, when APC are used to trigger chimeric receptors, the need for a flexible spacer between the scFv and TM domains becomes apparent. The CD8alpha-derived hinge successfully performs this task in chimeric scFv-Syk receptors regardless of its cysteine content. A cytotoxic T cell hybridoma expressing chimeric receptor genes composed of scFv-CD8(hinge)-CD8(TM)-Syk or scFv-CD8(hinge)-CD4(TM)-Syk is efficiently stimulated to produce IL-2 upon interaction with APC and specifically lyses appropriate target cells in a non-MHC-restricted manner.

Transfer of chimeric receptor gene made of variable regions of tumor-specific antibody confers anticarbohydrate specificity on T cells.

The antitumor specificity of T cells can be induced by gene transfer using a recently developed therapeutic approach (T body). In this work, we genetically conferred anticarbohydrate specificity onto T cells using the variable regions of monoclonal antibody MLuC1, which binds the Lewis(Y) (LeY) tumor-associated antigen that is overexpressed on several human carcinomas. The variable regions of MLuC1, which are in a single-chain Fv (ScFv) configuration, were cloned and spliced in a eukaryotic expression vector with both the gene encoding the signal-transducing gamma-chain of the human Fcgamma receptor and a flexible hinge domain. The chimeric ScFv-gamma gene was expressed in a murine cytotoxic T-cell hybridoma. Transfectants receiving vector only served as a negative control (mock). Screening for functional transfectants was carried out using a tumor growth inhibition assay. The soluble form of MLuC1 ScFv was recovered from bacteria periplasm and tested for binding to LeY-expressing cells by the fluorescence-activated cell sorter analysis. Despite the low binding ability of the soluble MLuC1 ScFv, 7 of 13 genetically engineered cytotoxic T lymphocyte clones inhibited the growth of LeY-positive cells and did not affect growth of LeY-negative cells. None of the mock clones tested specifically inhibited tumor growth. These data indicate that, by chimeric MLuC1 ScFv-gamma gene transfer, it is possible to confer anticarbohydrate specificity onto T cells and extend the applicability of the T-body approach to tumor-associated antigens that are naturally not recognized by T cells.

Tyrosine kinase chimeras for antigen-selective T-body therapy.

Protein tyrosine kinases (PTKs) transmit activation signals in almost every cell type, including immune effector cells. The aberrant or constitutive activation of PTKs can often cause neoplastic transformation. The use of chimeric receptors based on PTKs may enable us to elucidate the signaling pathways of normal immune cells and other cell types, and the abnormal events that can lead to malignant transformation. In this review, we focus on antigen specific chimeric PTKs in which antibody-derived scFv are joined to the Syk family of PTKs. These chimeric receptors yielded reagents that can selectively redirect immune effector cells and specifically activate them to produce cytokines or lyse their target. The advantages of using such PTK-based chimeras to redirect lymphocytes to tumor targets and their potential as an immunotherapeutic approach to malignant disease is discussed.

Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies.

The generation of catalytic antibodies should enable the catalysis of reactions for which no enzymatic or chemical catalyst is currently available. In previous studies, we established a series of catalytic antibodies capable of hydrolysing p-nitrobenzyl (pNB) and p-nitrophenyl (pNP) esters. A group of these catalytic antibodies exhibited high reactivity and substrate specificity, yet each individual antibody demonstrated different kinetic parameters. In order to study the molecular basis for these differences, we have cloned, sequenced and expressed the variable regions of this group of antibodies as functional scFv and Fv in bacteria. The variable region of the heavy chain is derived from a novel germline gene of the J558 family whereas the light chain comes from a germline gene previously found in our catalytic antibodies catalysing the hydrolysis of only nitrophenyl esters, demonstrating that the heavy chain determines the specificity for the nitrobenzyl esters. Several different expression systems were examined for their ability to produce catalytically active antibodies. When expressed as an scFv, both refolded and secreted scFvs exhibited catalytic activity although yields of expressed protein were low. The secreted scFvs had higher specific activity. On the other hand, Fv fragments were expressed in sufficient quantities to allow kinetic analysis. Levels of expression were dependent on the sequence of VL used. Using this expression system, the relative contributions of the individual light and heavy chains to catalysis and binding could be evaluated. Both original VH and VL regions are required for hapten binding, although the VH is more crucial for catalysis. By replacing the CDR3 of the heavy chain with a random sequence, it was shown to be essential for both binding and catalysis. This expression system together with site-directed mutagenesis should enable a more detailed study of the catalytic mechanism of this set of antibodies.

X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release.

The x-ray structures of the unliganded esterase-like catalytic antibody D2.3 and its complexes with a substrate analogue and with one of the reaction products are analyzed. Together with the structure of the phosphonate transition state analogue hapten complex, these crystal structures provide a complete description of the reaction pathway. At alkaline pH, D2.3 acts by preferential stabilization of the negatively charged oxyanion intermediate of the reaction that results from hydroxide attack on the substrate. A tyrosine residue plays a crucial role in catalysis: it activates the ester substrate and, together with an asparagine, it stabilizes the oxyanion intermediate. A canal allows facile diffusion of water molecules to the reaction center that is deeply buried in the structure. Residues bordering this canal provide targets for mutagenesis to introduce a general base in the vicinity of the reaction center.

Mechanism of inactivation of a catalytic antibody by p-nitrophenyl esters.

Antibody CNJ206 catalyses the hydrolysis of p-nitrophenyl esters with significant rate enhancement; however, after a few cycles, 90% of the catalytic activity of CNJ206 is irreversibly lost. This report investigates the properties of the inactivated Fab (fragment antigen binding). After inactivation, the residual esterase activity of CNJ206 is similar to that of the catalytic antibody inhibited by the transition-state analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered. Here we propose a simple scheme that accounts for the steady-state kinetics of inactivation. The following lines of evidence, when taken together, suggest that stable acylated tyrosine side chains within or close to the Fab combining site are involved in the inactivation process: isoelectric focusing and matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometry show that incubation with substrate results in several acylated Fab species; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, which is slowed down by the presence of the TSA hapten. Analysis of the Fab-TSA X-ray structure shows that three tyrosine residues are potential candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site-directed mutation of one or more of these residues might prevent substrate inactivation and significantly improve catalysis.

Efficient and selective p-nitrophenyl-ester-hydrolyzing antibodies elicited by a p-nitrobenzyl phosphonate hapten.

A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)-phosphonate transition-state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl-ester, were also found to catalyze the hydrolysis of the analogous p-nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np-ester is higher in terms of rates (k(cat); as expected from the higher intrinsic reactivity of Np-esters); however, the rate acceleration (k(cat)/k(uncat)) is close to or lower than that observed with the Nbzl-ester. Unexpectedly, the affinity to the Np-ester substrate (1/K(M)) and therefore k(cat)/K(M) are significantly higher. The best example is antibody D2.4 having a k(cat)/K(M) value of 64 s(-1) x M(-1) with the Nbzl-ester and 9400 s(-1) x M(-1) with the Np-ester. Moreover, due to a lower product inhibition by p-nitrophenol relative to p-nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np-ester. The differential affinity of these antibodies to the Nbzl-phosphonate TSA versus the Nbzl-ester substrate (K(S)/K(TSA) or K(M)/K(i)) correlates well with the observed rate enhancement (k(cat)/k(uncat)). For the Np-ester, however, stabilisation of the transition state (as reflected by K(S)/K(TSA) and by the catalytic proficiencies, k(cat)/K(M)/k(uncat)) does not fully account for the catalytic power (k(cat)/k(uncat)), indicating a more complex catalytic mechanism than simply transition-state stabilization. A comparison of the kinetic parameters of D2.4 with other Np-ester-hydrolyzing antibodies raised against Np-phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl-phosphonate hapten. These results appear to be general: anti-(Nbzl-phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np-esterases. They demonstrate the use of an expanded TSA-hapten, where a spacer (a methylene group) mimics bonds that are partially cleaved in the transition state of the catalyzed reaction.

Direct T cell activation by chimeric single chain Fv-Syk promotes Syk-Cbl association and Cbl phosphorylation.

The protein tyrosine kinase Syk is activated upon engagement of immune recognition receptors. We have focused on the identification of signaling elements immediately downstream to Syk in the pathway leading to T cell activation. To circumvent T cell receptor (TCR). CD3 activation of Src family kinases, we constructed a signaling molecule with an extracellular single chain Fv of an anti-TNP antibody, attached via a transmembrane region to Syk (scFv-Syk). In a murine T cell hybridoma, direct aggregation of chimeric Syk with antigen culminates in interleukin-2 production and target cell lysis. Initially, it causes an increase in the association between scFv-Syk and the cytosolic protein Cbl and subsequently promotes tyrosine phosphorylation of Cbl. Interestingly, although both Cbl and phospholipase C-gamma (PLC-gamma) are phosphorylated in this hybridoma upon TCR.CD3 cross-linking, these two events are uncoupled in scFv-Syk-transfected cells, in which we were unable to detect antigen-driven PLC-gamma phosphorylation. These results support a model in which Syk can initiate and directly activate the T cell's signaling machinery and position Cbl as a primary tyrosine kinase substrate in this pathway. Furthermore, for efficient PLC-gamma phosphorylation to occur in these cells, the combined actions of different tyrosine kinase families may be required.